Colistin Resistance in Acinetobacter baumannii: Basic and Clinical Insights

Abstract

The emergence of drug-resistant bacteria has posed a significant problem in medical institutions worldwide. Colistin, which targets lipopolysaccharide (LPS), serves as a last-resort antimicrobial agent against multidrug-resistant Gram-negative bacteria. Nevertheless, Acinetobacter baumannii, a pathogen with a worldwide prevalence of antimicrobial resistance, has been reported to develop resistance to colistin frequently. In this review, we discuss how A. baumannii acquires resistance to colistin, focusing on modification as well as loss of LPS present in its outer membrane, which is the primary mechanism of A. baumannii’s resistance to colistin. Basic and clinical insights regarding colistin resistance in A. baumannii have been discussed in isolation. Therefore, we discuss the relationship between these 2 colistin resistance mechanisms in terms of the frequency and fitness of genetic mutations based on the insights from basic studies and clinical settings. We concluded that understanding the detailed mechanisms of colistin drug resistance requires a comprehensive understanding of both the frequency of mutations and the effects of selection pressure. Finally, we highlight the importance of promoting research from both basic science and clinical perspectives.

1. INTRODUCTION

Acinetobacter spp. are aerobic Gram-negative bacilli broadly distributed in nature.¹⁾ These bacteria can survive in harsh environments, withstanding dryness, humidity, and nutrient depletion. Acinetobacter baumannii is especially problematic, often causing nosocomial infections.^1–5) It is a typical opportunistic pathogen, causing diverse infections such as ventilator-associated pneumonia and bloodstream infections including sepsis, urinary tract infections, and wound infections in immunocompromised hosts.^5–9) A. baumannii has a high capacity to acquire a broad spectrum of antimicrobial resistance (AMR); thereby, the emergence of carbapenem-resistant A. baumannii has become a significant issue in healthcare facilities worldwide.^10–13) Most carbapenem-resistant bacteria are resistant to several other drugs as well, resulting in difficulties in treating infections caused by these organisms. Multidrug-resistant A. baumannii is one of the main bacteria associated with AMR and is graded at the top of the Priority Pathogen List announced by WHO in 2017, Center for Disease Control and Prevention in 2019, and Japan Agency for Medical Research and Development in 2021, signaling an urgent need for new antimicrobial agents.^14–17)

Colistin (polymyxin E), a cyclic cationic decapeptide antibiotic (Fig. 1A), exhibiting robust antibacterial activity against Gram-negative bacteria, was isolated from Bacillus colistinus by Koyama in 1950.¹⁸⁾ Following the development of the sulfate and hydrochloride salts, a low-toxicity derivative, the sodium methanesulfonate salt formulation, was launched as an injectable formulation. Upon intravenous administration, injectable colistin methanesulfonate is hydrolyzed to colistin in vivo, which exhibits antibacterial activity.^19,20) Colistin was widely used as a treatment for Gram-negative bacterial infections until the 1970s. However, due to its association with a high incidence of renal dysfunction and neurotoxicity, and the development of safer alternatives like β-lactams and aminoglycosides, colistin was curtailed, and its approval as a drug was withdrawn.^21–25) Recently, the emergence of multidrug-resistant Gram-negative bacteria, coupled with stagnation in developing new antimicrobial agents, has caused global concern. Therefore, WHO positioned colistin as an important antimicrobial agent in clinical practice in 2012,²⁶⁾ and it is once again attracting attention. Japan reapproved the use of colistin in 2015 to treat severe infections caused by multidrug-resistant Gram-negative bacteria. Currently, colistin is used as a “last resort” in clinical practice against drug-resistant bacteria, such as carbapenem-resistant A. baumannii, against which limited effective drugs are available.^22,27–30) Colistin is recommended to be used in combination with carbapenems, rifampicin, and other antimicrobial agents to prevent the development of bacterial resistance, improve efficacy, mitigate side effects, and improve pharmacokinetics.^24,31,32)

Fig. 1. The Structure (A) and Mechanism of Bactericidal Action (B) of Colistin, a Cyclic Cationic Decapeptide Antibiotic

Colistin destabilizes the outer membrane of bacteria through electrostatic interaction with anionic lipopolysaccharides, resulting in a bactericidal action.

The cationic decapeptide of colistin (Fig. 1A) destabilizes the outer membrane of Gram-negative bacteria through electrostatic interaction with anionic lipopolysaccharides (LPS) in the outer membrane. It induces local damage to the outer membrane by replacing the LPS-stabilizing calcium (Ca²⁺) and magnesium (Mg²⁺) ions.^20,24,30,33) These mechanisms increase the permeability of the bacterial cell membrane, causing intracellular substances to leak out of the cell, resulting in a bactericidal action (Fig. 1B). In other antibacterial mechanisms of colistin, bacterial cell death is induced by hydroxyl radical generation and inhibition of respiratory chain enzymes.^25,34)

Acinetobacter baumannii has been reported to develop resistance to colistin frequently. Many colistin-resistant A. baumannii strains arise through alterations in its LPS. Two major mechanisms have been proposed for resistance to colistin: modification of LPS by regulating PmrAB, which is also observed in other bacterial species such as E. coli,^35,36) and loss of LPS by mutation of lpxACD genes, which is an A. baumannii-specific mechanism (Fig. 2). Until now, the basic research and clinical findings on these 2 colistin resistance mechanisms in A. baumannii have been discussed independently. This review explores colistin resistance mechanisms involving the modification and loss of LPS in A. baumannii from both basic and clinical perspectives.

Fig. 2. The Mechanism of Colistin Resistance in Acinetobacter baumannii Caused by LPS Modification (Left Panel) or Deficiency (Right Panel)

LPS modification is regulated by PmrAB, and the phosphate groups at positions C1′ and/or C4′ of lipid A are modified by phosphorylethanolamine (pEtN) or galactosamine (GalN). Other mechanisms include activation of eptA by ISAba1 and acquisition of mcr by plasmid (left panel). LPS deficiency is caused by mutations in the genes encoding LpxACD, which are involved in the biosynthesis of lipid A (right panel). LPS, lipopolysaccharide.

2. UNDERSTANDING THE MECHANISM OF COLISTIN RESISTANCE: LIPOPOLYSACCHARIDE MODIFICATION AND DEFICIENCY

PmrAB is a 2-component system in bacteria; the sensor kinase PmrB in the inner membrane responds to specific environmental factors, and this response regulates the expression of various genes, including those encoding LPS-modification enzymes (such as PmrC) through the response regulator PmrA.^36–38) Adams et al. first reported that activating mutations in pmrAB promote the expression of the pmrCAB operon, resulting in the acquisition of colistin resistance via LPS modification.³⁹⁾ Subsequently, a number of pmrAB mutants have been identified from colistin-resistant clinical isolates,^40–45) and from patients receiving colistin therapy.^45,46) LPS modification targets phosphate in the C1′ and/or C4′ region of lipid A in the LPS structure.⁴⁷⁾ Both phosphoethanolamine (pEtN)^45,47,48) and galactosamine (GalN)^47,49) have been identified as potential modification molecules (Fig. 2, left panel). In our recent study, we found that PmrAB responds to low pH, Fe²⁺, Zn²⁺, and Al³⁺ and that modifying the pEtN to LPS in response to these metal ions makes it resistant to colistin.⁵⁰⁾ LPS modification neutralizes the anionic charge, disrupting the interaction with colistin, resulting in resistance.

LPS modification mechanisms that are independent of the 2-component system are also known (Fig. 2, left panel). Clinical isolates of colistin-resistant strains have been identified, in which LPS modification was triggered by insertion of the insertion sequence ISAba1, which has promoter activity, upstream of eptA (a homolog of pmrC).^51,52) A plasmid colistin-resistant gene mcr, encoding a pEtN modification enzyme in E. coli, was identified in 2015.⁵³⁾ Recently, A. baumannii strains carrying plasmid-derived mcr genes have been reported. Currently, both mcr-1 and mcr-4.3 have been detected in A. baumannii.^54,55) There are concerns about the spread of colistin resistance through horizontal gene transmission. Thus, colistin resistance is attributed to the chromosomal and the plasmid-based LPS modification genes.

Although LPS is considered vital for the survival of Gram-negative bacteria, A. baumannii has the unique ability to survive without LPS. LPS deficiency is a very rare biological phenomenon and has been reported only in Neisseria meningitidis,⁵⁶⁾ Moraxella catarrhalis,⁵⁷⁾ and Acinetobacter nosocomialis.⁵⁸⁾ Moffat et al. first reported that LPS deficiency in A. baumannii resulted in the development of colistin resistance.⁵⁹⁾ Mutations in the lpxA, C, and D genes, which encode lipid A biosynthetic enzymes, cause this deficiency^60–63) (Fig. 2, right panel).

How does only A. baumannii possess the capacity to induce LPS deficiency? Some strains of A. baumannii can survive without LPS, while others cannot. Deletion of PBP1A, a penicillin-binding protein that forms the peptidoglycan layer, in A. baumannii strains, which do not survive without LPS, enables them to do so.⁶⁴⁾ In addition, peptidoglycan synthesis by the elongasome and a peptidoglycan-recycling enzyme were vital for LPS-deficient strains.⁶⁵⁾ These results suggest that survival of LPS-deficient strains likely involves structural changes in the peptidoglycan layer, to the extent that it affects cell morphology. Although LPS-deficient strains show significantly reduced proliferative ability compared to wild-type strains, reports indicate that this ability can be restored by passage in a liquid medium. Restoration of proliferative ability results from decreased expression of pldA and mla, which encode enzymes that repair the outer membrane lipid conformation.^66,67) Strains deficient in these genes demonstrate improved proliferative ability, suggesting that LPS-deficient strains might undergo significant remodeling of outer membrane lipids. However, the definitive factors enabling LPS deficiency in A. baumannii remain unknown.

3. OTHER MECHANISMS OF COLISTIN RESISTANCE BEYOND LPS-MODIFICATION/DEFICIENCY

Park et al. discovered that an activating mutation in pmrAB enhances the production of outer membrane vesicles (OMVs).⁶⁸⁾ These overproduced OMVs acted as decoys to protect themselves from polymyxin B (an antibacterial drug similar to colistin) and also contributed to protect surrounding bacteria. Thus, mutations in pmrAB contribute to colistin resistance not only through LPS modification but also via OMV production.

Other studies have reported that the loss of LpsB, a glycosyltransferase responsible for synthesizing LPS core polysaccharides, results in increased sensitivity to both colistin and cationic host defense peptides.⁶⁹⁾ In addition, amino acid substitution mutations in LpsB (*241K, H181Y) and mutations in lpxACD and pmrAB were detected simultaneously in clinical isolates of colistin-resistant strains, suggesting a connection to colistin resistance.^70,71) Deletion of LptD, which plays a role in the transport of LPS from the cytoplasm to the outer membrane, also results in colistin resistance.⁷²⁾ Hence, protein mutations also related to the structure and localization of LPS are implicated in colistin resistance.

In A. baumannii, the drug efflux pump system also plays a crucial role in colistin resistance. Lin et al. used an emrB deletion mutant and demonstrated that colistin resistance in A. baumannii decreases, suggesting that the EmrAB efflux system contributes to resistance.⁷³⁾ Furthermore, colistin-resistant strains showed a decrease in resistance when treated with the efflux pump inhibitor cyanide-3-chlorophenylhydrazone.^74,75)

Additionally, it has been reported that colistin resistance in A. baumannii increases due to genetic deficiency of the deacetylase PgaB, one of the biosynthetic enzymes of the extracellular polysaccharide poly-β-1,6-N-acetyl-glucosamine (PNAG), a component of the biofilm.⁷⁶⁾ It is suggested that colistin targets not only the LPS in the outer membrane but also that in the inner membrane.⁷⁷⁾ Therefore, the deficiency of PgaB, leading to the accumulation of PNAG in the periplasmic region, thus preventing colistin from binding to LPS in the inner membrane, may contribute to resistance.

4. BASIC CONSIDERATIONS ON THE MECHANISMS OF COLISTIN RESISTANCE IN A. BAUMANNII ISOLATED FROM CLINICAL SETTINGS

As described above, 2 primary mechanisms of colistin resistance exist in A. baumannii. LPS-deficient strains result from mutations that cause a decline in or loss of function of any of the 3 enzymes, LpxA, C, or D (Fig. 2, right panel), while LPS-modified strains result from activating mutations in PmrAB (Fig. 2, left panel). Protein loss-of-function mutations occur not only due to missense or nonsense mutations but also due to the insertion or deletion of nucleotides that lead to frameshift mutations. Insertion of insertion sequences such as ISAba1 and ISAba11 into the lpxACD gene has also been reported to cause loss-of-function mutations.^78,79) By contrast, gain-of-function mutations in PmrAB, occurring through missense mutations, cause specific amino acid changes. In cases where the frequency of genetic mutation is constant, loss-of-function mutations, resulting in LPS-deficient strains, are expected to occur more frequently than gain-of-function mutations, resulting in LPS-modified strains (Fig. 3, center column). Several colistin-resistant strains, established in basic research using colistin alone for selection, were LPS deficient.⁶⁰⁾ However, most colistin-resistant strains isolated from clinical settings were LPS modified.^{32,43,60,80,81)}

Fig. 3. Schematic Diagram of the Emergence of Colistin-Resistant Acinetobacter baumannii Strains and Their Selection Pressure

There is diversity in the population and a certain number of mutant strains (left column). In basic experiments, LPS-deficient strains with a high frequency of mutations are selected for colistin (center column). In clinical settings, alongside high fitness LPS-modified strains are selected due to additional selective pressures such as other antibiotics and host immunity (right column). LPS, lipopolysaccharide.

Deletion of LPS in A. baumannii increases resistance to colistin but increases the susceptibility to β-lactams, quinolones, aminoglycosides, and some disinfectants.^60–62) In clinical practice, appropriate antimicrobial therapy and prevention using disinfectants are practiced against A. baumannii infections, and LPS-deficient strains are likely to be eliminated by these antimicrobial agents and disinfectants even if these do appear. In addition, colistin therapy is aggressively used in combination with other antimicrobial agents to improve efficacy and prevent the emergence of resistant strains. The combination of colistin and carbapenems exerts synergistic effects against carbapenem-resistant A. baumannii strains in vitro and is used in clinical practice.^82–87) However, LPS-modified strains emerged during the treatment of carbapenem-resistant A. baumannii when colistin and other antimicrobial agents were used⁸⁸⁾; therefore, caution should be exercised when these are used in combination. Furthermore, in clinical trials, combination therapy with colistin and meropenem against carbapenem-resistant A. baumannii strains increased mortality compared to that when colistin alone was used,^89,90) indicating a significant discrepancy between in vitro and in vivo results. In our basic study, the frequency of colistin-resistant strains decreased when colistin and sub-MIC meropenem were used in combination for selection, whereas only LPS-modified strains were established when colistin alone was used for selection.⁶⁰⁾ Therefore, it is essential to provide antimicrobial therapy at appropriate, effective concentrations to prevent the emergence of drug-resistant strains, and the pharmacist’s role in AMR countermeasures is considered significant.

LPS-deficient A. baumannii strains have lower proliferative ability and higher fitness costs than the wild-type strains.^{60,62,91–93)} In addition, LPS-deficient strains reduce resistance to antimicrobial peptides such as lysozyme, produced by neutrophils of host immune cells.^61,94) By contrast, LPS-modified strains do not exhibit decreased proliferative ability; neither do these exhibit significantly increased susceptibility to antimicrobial agents other than colistin.^60,94) Furthermore, studies evaluating the host lethality of LPS-deficient and LPS-modified strains showed that LPS-modified strains were lethal, while LPS-deficient strains were barely lethal.^92,94) These results suggest that LPS-deficient strains are less virulent than LPS-modified strains and are more easily eliminated from the host. In clinical settings, LPS-deficient strains are likely to be weeded out by antimicrobial agents, disinfectants, and selection pressure from the host. Therefore, A. baumannii colistin-resistant strains isolated from clinical practice are mostly LPS-modified strains (Fig. 3, right column). Thus, it is crucial to comprehensively consider both the frequency of mutation and the effect of selection pressure to understand the drug resistance mechanism of bacteria.

Karakonstantis reported in a systematic review that colistin-resistant A. baumannii strains that appear in vivo are mainly LPS modified,⁹⁵⁾ which should be noted clinically. Consequently, studies on the 2-component system PmrAB, which is responsible for LPS modification, need to be promoted. Currently, studies on PmrAB in A. baumannii are mainly based on analyses using clinical isolates, which are difficult to compare because of their different genetic backgrounds, limiting our understanding. Some reports indicate that the pathogenicity and fitness costs of LPS-modified strains are not different from those of wild-type strains.^59,96,97) By contrast, other reports indicate that both pathogenicity and fitness are reduced in LPS-modified strains.^92,93,98) Furthermore, certain studies reported that LPS-modified strains modified by GalN are observed in PmrAB-mutant clinical isolates and show resistance to colistin⁴⁹⁾ (Fig. 2, left panel), whereas other studies reported that modification by GalN is not observed.⁹⁹⁾ We succeeded in constructing a library of LPS-modified strains with mutations in PmrAB using colistin in combination with other antimicrobials and selecting colistin-resistant strains of the same genetic background as wild-type strains.⁶⁰⁾ Using this library, we expect to reveal the regulon and environmental response factors regulated by PmrAB in A. baumannii, which have not yet been elucidated. This could lead to a detailed phenotypic understanding of LPS-modified strains.

5. SIGNIFICANCE OF BASIC RESEARCH IN UNDERSTANDING COLISTIN RESISTANCE OF A. BAUMANNII

Analyzing the mechanism of LPS deficiency will help us understand the significance of LPS in bacteria, and the adaptive strategies employed by bacteria for survival. In addition to LPS deficiency and modification, changes in core polysaccharide and LPS localization have been reported in some colistin-resistant A. baumannii strains.^69–72,100) These mutant strains can be applied as useful analytical tools for understanding the biological role of LPS. Recently, an innovative approach was reported in a study in which re-sensitization to colistin was achieved by inhibiting the enzymatic activity of the pEtN-modifying enzyme with a silver ion.¹⁰¹⁾ Thus, it is expected that basic research will lead to the development of novel therapeutic and control strategies for colistin-resistant strains.

One of the interesting phenotypes of LPS-deficient strains is that the presence of colistin, rather than the absence, promotes proliferative activity. Furthermore, colistin-dependent strains, which can survive only in the presence of colistin, have emerged both in the laboratory and in clinical settings.^{100,102–105)} Colistin-dependent strains are explained by a mutation in catalase KatG, which is responsible for removing reactive oxygen species.¹⁰⁶⁾ In the presence of colistin, it stabilizes the LPS-deficient outer membrane and inhibits the influx of reactive oxygen species, whereas in the absence of colistin, the influx of reactive oxygen species cannot be suppressed. Mutations in KatG induce cell death due to the inability to remove reactive oxygen species. Colistin-dependent strains are a factor that determines the success rate of colistin therapy.¹⁰⁵⁾ Thus, these are interesting biological phenomena in which bacteria use antimicrobial agents for their own survival, even though these agents are designed to kill bacteria. An important consideration is that colistin-dependent strains do not form colonies on regular agar media, making these undetectable as clinical isolates despite their existence.

Heteroresistance is a growing concern in the field of antimicrobial-resistant infections.^107,108) Although the definition of heteroresistance differs among studies, the term “heteroresistance” is broadly used to describe a phenomenon in which the subpopulation of seemingly isogenic bacteria exhibit wide-range susceptibility to a particular antibiotic.¹⁰⁸⁾ Heteroresistance to colistin has also been reported in A. baumannii.^{100,109–111)} It has been observed that the population of LPS-modified strains that are dominant during colistin-therapy in clinical settings decreases by the end of colistin treatment and the LPS-modified strains are replaced by the wild-type strains.¹¹²⁾ A systematic review of colistin-heteroresistance in Acinetobacter spp. showed that the prevalence of heteroresistance was 33%, and, in almost all cases, a heterogenous population, mainly composed of wild-type strains and pmrAB or lpxACD mutants, was observed.¹¹³⁾ Persister cells, which comprise a small number of cells in a large population of genetically colistin-susceptible or -resistant strains, may be important for heteroresistance.^110,114) A few of the 85 colistin-resistant strains that were established in our previous study⁶⁰⁾ were resistant to colistin despite being genetically identical to the wild type (unpublished observation). Populations exhibiting heteroresistance to colistin may arise based on the frequency and fitness of LPS-deficient and -modified strains.

6. CONCLUSION

In clinical practice, LPS-modified A. baumannii colistin-resistant strains are frequently generated due to selective pressure from antimicrobial agents, disinfectants, and the host. However, LPS-deficient A. baumannii strains may also frequently emerge in this process, leading to diversity. To understand the complex mechanism of colistin resistance in detail, it is essential to conduct basic research that includes LPS-modified as well as -deficient strains. To confront the problem of AMR, including the development of colistin resistance, it is important to promote research from both basic and clinical perspectives (Fig. 3).

Acknowledgments

We are grateful to Dr. Norihiko Takemoto (National Center for Global Health and Medicine) for helpful discussions, and Mr. Yuichi Nakamura for preparing the illustrations.

Conflict of Interest

The authors declare no conflict of interest.

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